May 02, 2022
Preparation of BSA complexed free fatty acids for in vitro studies
- 1Montreal Diabetes Research Center, CRCHUM, Montréal, QC, Canada.;
- 2Montreal Diabetes Research Center, CRCHUM, and Department of Medicine, Université de Montréal, Montréal, QC, Canada.
Protocol Citation: Caroline CT Tremblay, Julien Ghislain, Vincent Poitout 2022. Preparation of BSA complexed free fatty acids for in vitro studies. protocols.io https://dx.doi.org/10.17504/protocols.io.b6r2rd8e
Manuscript citation:
Briaud I, Harmon JS, Kelpe CL, Segu VB, Poitout V. Lipotoxicity of the pancreatic beta-cell is associated with glucose-dependent esterification of fatty acids into neutral lipids. Diabetes. 2001 Feb;50(2):315-21. doi:10.2337/diabetes.50.2.315. PMID: 11272142.
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: March 25, 2022
Last Modified: May 02, 2022
Protocol Integer ID: 59930
Keywords: fatty acids, BSA complex, cell culture
Funders Acknowledgement:
CIHR
Grant ID: 0406014428
NIH
Grant ID: 5R01DK058096-15
Abstract
This protocol describes the steps to prepare a fatty acid:BSA complex in cell culture media. It is suitable for the treatment of primary tissue (eg. isolated islets) and cell lines in ex vivo culture. Briefly, fatty acid stock solutions are prepared and complexed with BSA prior to dilution in culture media. Final fatty acid concentrations range from 0.125 to 0.5 mM. We recommend replacing the fatty acid mixture once each day throughout the treatment.
Materials
MilliQ Water
Ethanol, AnhydrousBio Basic Inc.Catalog #D0193
Steriflip-GP Sterile Centrifuge Tube Top Filter UnitMilliporeCatalog #SCGP00525
sodium oleateMillipore SigmaCatalog #O7501
Sodium palmitateMillipore SigmaCatalog #P9767
Fatty Acid Free Heat Shock Bovine Serum Albumin Powderequitech bio, inc.Catalog #BAH66-0500
Preparation of BSA:FA complex
Preparation of BSA:FA complex
1h 25m
1h 25m
Preparation of Palmitate (PA) and Oleate (OA) stock solutions
As PA and OA are in powder form and very hydrostatic, handle with care as the powder will stick to everything and move about readily.
Carefully weigh out a sufficient amount of each to make 150 millimolar (mM) stock solutions in ethanol.
MW (PA) = 278.41, stored in a desiccator at 4 °C .
MW (OA) = 305, stored in a desiccator at -20 °C .
Preparation of 1 mL of 150 millimolar (mM) stock solution:
PA Weight out 41.8 mg and put in a 1.5 ml tube.
Add 1 mL of 50 % (v/v) ethanol.
Vortex and heat at65 °C until dissolved; vortex periodically.
OA Weight out 45.7 mg and put in a 1.5 ml tube.
Add 500 µL of 100 % (v/v) ethanol.
Vortex and heat at 65 °C for 00:15:00 ; mix occasionally.
Then add 500 µL of MilliQ water, and vortex. The remaining powder should dissolve instantly.
Return to 65 °C until ready for use.
15m
Preparation of 0.5 mM fatty acid solution in culture media (FA:BSA at 5:1 molar ratio)
Prepare 10 % (v/v) fatty acid-free BSA:
Weight 5 g and dissolve in50 mL of MilliQ water.
Filter solution with a Millipore Steriflip-GP Sterile Centrifuge Tube Top Filter Unit.
Store at 4 °C .
Under a laminar flow hood, add the following components per ml of culture media:
Place 67 µL of 10 % (v/v) fatty acid-free BSA into a 15 ml Falcon tube.
Heat in 37 °C water bath for 00:05:00 .
Add 3.3 µL of the 150 millimolar (mM) fatty acid stock solution.
Return to the 37 °C water bath for 00:05:00 . Visually check for cloudiness. If clear, great, if cloudy, start over.
Incubate in the 37 °C water bath for 01:00:00 .
Then add 930 µL of culture medium (eg. RPMI 1640 plus 10 % (v/v) FBS) heated to 37 °C .
For control tube (Vehicle/BSA), add 3.3 µL of 50 % (v/v) ethanol to 67 µL of 10 % (v/v) fatty acid-free BSA into a 15 ml Falcon tube, and treat as above.
1h 10m
Preparation of 0.15 mM fatty acid solution in culture media (FA:BSA at 1.5:1 molar ratio)
To prepare a solution with a final fatty acid concentration of 0.125 millimolar (mM) proceed as described in step 2 adding 1 µL of the 150 millimolar (mM) fatty acid stock solution (instead of 3.3 µL ). Also reduce the ethanol to 1 µL for the control.
To prepare any fatty acid concentration up to0.5 millimolar (mM) final adjust the amount of fatty acid stock solution accordingly while keeping the BSA constant.